Inward rectifier K (Kir) channels are essential for the normal function of both excitable and nonexcitable cells. The specific aims of this proposal are two-fold: to better understand the mechanisms of inward strong rectification in Kir2.1 (IRK1), the major component of the high resting K conductance in many cell types, and to investigate the basis of mechanosensitivity of the G-protein regulated K channel Kir3.4 (GIRK4), a major component of the resting K conductance in atrial muscle and brain. We will apply electrophysiological (patch-clamp), molecular biological and biochemical techniques to various cloned Kir channels expressed in Xenopus oocytes or mammalian cell lines. In the first specific aim, we will determine how an intrinsic gating mechanism, which we have recently identified and postulate to be a tethered gating particle, interacts with polyamines and Mg to contribute to strong inward reactivation in Kir channels. We will test the novel hypothesis that the tethered gating particle contains binding sites for polyamines and Mg which enhances its ability to cause inward rectification, providing further insight into the molecular basis of strong inward rectification. In the second specific aim, we will characterize the molecular mechanisms underlying stretch-induced inactivation of Kir3.x channels, a property which we have recently identified in Kir3.4 and native cardiac KACh channels. We will determine: whether mechanosensitivity is also a property of other members of the Kir3.x family, the regions of the Kir3.4 channel required for mechanosensitivity, using chimeric constructs and site-directed mutagenesis; the role of G proteins; and the cytoskeletal and/or extracellular matrix elements responsible for transducing mechanosensitivity. The mechanosensitivity of Kir3.x channels may contribute to a variety of stretch-induced responses, including stretch- induced arrhythmias, atrial natriuretic peptide (ANP) release, and/or hypertrophic gene programming. Together, these studies in Kir channels will provide important insights into the regulation of excitability in ventricular and atrial cardiac muscle, as well as in other excitable tissues.